Compositions and methods that modulate bacteria in a companion animal

ABSTRACT

A specific bacterium in a companion animal can be improved by adjusting the diet of the animal to increase the amount of a compound which positively or negatively modulates the specific type of bacterium or to decrease the amount of a compound which positively or negatively modulates the specific type of bacterium. The specific type of bacterium can be one or more of  Bifidobacterium, Lactobacillus , or  Clostridium perfringens.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/434,560 filed Dec. 15, 2016, the disclosure of which isincorporated in its entirety herein by this reference.

BACKGROUND

Probiotics have been used to modulate the microflora in animals as wellas confer various health benefits to the animal. Probiotics can bedefined as live microorganisms that confer a health benefit to a hostwhen administered in adequate amounts. It is theorized that probioticsmay impart their beneficial health effects either by increasing theresistance to colonization of mucosal surfaces by pathogenic bacteria(colonization resistance) or by exerting a direct effect on gutassociated lymphoid tissue (GALT) that promotes the production ofimmunomodulators.

Probiotics have been used to modulate the course of a variety ofinfectious diseases in human medicine. In contrast, few studies havebeen performed in veterinary medicine. The majority of veterinarystudies have been in large animals where probiotics have been used toattempt to alter the shedding of fecal pathogens or to improveproduction parameters such as weight gain, feed conversion rate, andreduced mortality. While benefits can be conferred, the addition ofprobiotics can provide unintentionally consequences including digestivediscomfort, over stimulation of the immune system, and gene transfer.

SUMMARY

The present disclosure relates generally to pet food compositions;methods of enhancing nutritional benefit of a pet food; and methods formodulating bacteria of at least one of Bifidobacterium, Lactobacillus,or Clostridium perfringens in a companion animal. Specifically, thepresent disclosure relates to metabolites for modulating at least one ofBifidobacterium, Lactobacillus, or Clostridium perfringens in acompanion animal.

The present inventors have developed a predictive model ofBifidobacterium, Lactobacillus, and Clostridium perfringens byidentifying metabolite compounds which correlate to Bifidobacterium,Lactobacillus, or Clostridium perfringens. A very controlled study wasemployed to minimize other external factors by using multiple caninesall fed the same diet. A validation model was then developed by feedingdifferent levels of the identified compounds (via a dietary change) to agroup of canines and measuring changes in the corresponding bacterium.

Accordingly, in a general embodiment, the present disclosure provides apet food composition comprising: protein, carbohydrates, fat, fiber, anda metabolite for modulating at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens in a companion animal. In oneembodiment, the pet food composition can provide at least a 5% increaseor a 5% decrease in the amount of Bifidobacterium, Lactobacillus, orClostridium perfringens present in the companion animal. In one aspect,the pet food can provide an increase. In another aspect, the pet foodcan comprise a decrease.

The present disclosure also provides methods of minimizing costsassociated with production of a pet food; methods of enhancingnutritional benefit of a pet food; methods for modulating the amount ofBifidobacterium, Lactobacillus, or Clostridium perfringens in acompanion animal; and methods of measuring a change in the amount of atleast one of Bifidobacterium, Lactobacillus, or Clostridium perfringensin a companion animal.

An advantage of one or more embodiments provided by the presentdisclosure is to increase or decrease Bifidobacterium, Lactobacillus, orClostridium perfringens in a companion animal by adjusting the diet ofthe animal to increase the amount of a compound which positivelymodulates the specific bacterium or to decrease the amount of a compoundwhich negatively modulates the specific bacterium.

Additional features and advantages are described herein and will beapparent from the following detailed description.

DETAILED DESCRIPTION Definitions

As used in this disclosure and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition” or“the composition” includes two or more compositions. The term “and/or”used in the context of “X and/or Y” should be interpreted as “X,” or“Y,” or “X and Y.” Where used herein, the terms “example” and “such as,”particularly when followed by a listing of terms, are merely exemplaryand illustrative, and are not exclusive or comprehensive.

As used herein, “about” is understood to refer to numbers in a range ofnumerals, for example the range of −10% to +10% of the referencednumber, within −5% to +5% of the referenced number, or in one aspect,within −1% to +1% of the referenced number, and in a specific aspect,within −0.1% to +0.1% of the referenced number. Furthermore, allnumerical ranges herein should be understood to include all integers,whole or fractions, within the range. Moreover, these numerical rangesshould be construed as providing support for a claim directed to anynumber or subset of numbers in that range. For example, a disclosure offrom 1 to 10 should be construed as supporting a range of from 1 to 8,from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and soforth.

All percentages expressed herein are by weight of the total weight ofthe food composition unless expressed otherwise. When reference is madeto the pH, values correspond to pH measured at 25° C. with standardequipment. An “amount” can be the total amount of the referencedcomponent per serving of the composition or per distinct unit of thecomposition and/or can be the weight percentage of the referencedcomponent by dry weight. Moreover, an “amount” includes zero; forexample, the recitation of an amount of a compound does not necessarilymean that the compound is present, unless followed by a range thatexcludes zero.

The terms “food,” “food product” and “food composition” mean a productor composition that is intended for ingestion by an animal, including ahuman, and provides at least one nutrient to the animal. Further in thisregard, these terms mean that the product or composition is in a formready for consumption and is not merely an intermediate from which aconsumable product or composition is made, although other foodcompositions can be added in some embodiments. The term “pet food” meansany food composition intended to be consumed by a pet. The term “pet”means any animal which could benefit from or enjoy the compositionsprovided by the present disclosure. For example, the pet can be anavian, bovine, canine, equine, feline, hircine, lupine, murine, ovine,or porcine animal, but the pet can be any suitable animal.

The term “companion animal” means a dog or a cat. In an embodiment, thecompositions and methods disclosed herein involve a senior dog or asenior cat. Dogs are considered senior in the last 25% of their lives.The life span of a dog depends on its size and/or its breed, but for thepresent disclosure a senior dog is a dog that is at least 5 years of age(e.g., at least 6 years of age, at least 7 years of age, or at least 8years of age). The life span of a cat also depends on its size and/orits breed, but for the present disclosure a senior cat is a cat that isat least 7 years of age (e.g., at least 8 years of age, at least 9 yearsof age, or at least 10 years of age).

As used herein, “comparable companion animal” refers to a healthy animalof the same gender, breed, and age as the companion animal.

As used herein, “metabolite” refers to a compound having biologicalactivity in a companion animal that is an intermediate or product ofmetabolism, and includes precursors thereof. As used herein, “precursor”refers to any compound that metabolizes to a metabolite duringmetabolism in a companion animal. For example, if the specificmetabolite is cysteine, “the metabolite” comprises a cysteine precursor(e.g., methionine).

The term “effective amount” means an amount of a compound of the presentinvention that (i) treats or prevents the particular disease, condition,or disorder, (ii) attenuates, ameliorates, or eliminates one or moresymptoms of the particular disease, condition, or disorder, or (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition, or disorder described herein. In one embodiment, thepresent metabolite or combination of metabolites can be present in aneffective amount for modulating the amount of Bifidobacterium,Lactobacillus, or Clostridium perfringens in a companion animal.

The dosages expressed herein are in milligrams per kilogram of bodyweight per day (mg/kg/day) unless expressed otherwise.

The term “long-term administration” means periods of repeatedadministration or consumption in excess of one month. Periods of longerthan two, three, or four months can be used for certain embodiments.Also, more extended periods can be used that include longer than 5, 6,7, 8, 9, or 10 months. Periods in excess of 11 months or 1 year can alsobe used. Longer term use extending over 1, 2, 3, or more years areincluded in the invention. For certain aging animals, the animal willcontinue consuming on a regular basis for the remainder of its life.This can also be referred to as consumption for “extended” periods.

The term “regular basis” or “regular administration” means at leastmonthly dosing with the compositions or consumption of the compositions,and in one aspect, means at least weekly dosing. More frequent dosing orconsumption, such as twice or three times weekly, can be performed incertain embodiments. Still, in other embodiments, regimens can be usedthat comprise at least once daily consumption. The skilled artisan willappreciate that the blood level of a compound or certain metabolites ofthat compound or which result after the consumption of that compound,may be a useful tool for assessing or determining dosing frequency. Forexample, for determining feeding amounts for pet food compositionscomprising a certain metabolite, the blood concentration of thatmetabolite, may provide useful information. A frequency, regardless ofwhether expressly exemplified herein, that allows maintenance of adesired blood level of the measured compound, such as a metabolite,within acceptable ranges can be useful herein. The skilled artisan willappreciate that feeding amounts will be a function of the compositionthat is being consumed or administered as well as the animal consumingthe food, and some food compositions may require more or less frequentadministration to maintain a desired blood level of the measuredcompound (e.g., a metabolite).

The relative terms “improve,” “increase,” “enhance,” “decrease” and thelike refer to the effects of the composition disclosed herein (acomposition comprising a metabolites) relative to a composition having alower amount or lacking such metabolites, but otherwise identical.

A “blended” composition merely has at least two components having atleast one different characteristic relative to each other. In oneaspect, moisture content and water activity can be different in thecontext of the present disclosure. In this regard, description of acomposition as “blended” does not imply that the blended composition hasbeen subjected to processing sometimes referenced as “blending,” namelymixing components so that they are indistinguishable from each other,and, in one aspect, such processing is avoided when mixing one componentwith the other components to form a blended composition (e.g., mixing adry component with a wet or semi-moist component). Further in thisregard, in a blended composition each of the at least two componentshaving at least one different characteristic relative to each other canretain their distinct identity and appearance.

“Wet food” means a pet food having a moisture content from about 50% toabout 90%, and in one aspect, from about 70% to about 90%. “Dry food”means a pet food having a moisture content less than about 20%, and inone aspect, less than about 15%, and in a specific aspect, less thanabout 10%. “Semi-moist food” means a pet food having a moisture contentfrom about 20% to about 50%, and in one aspect, from about 25% to about35%.

“Kibbles” is used synonymously with “chunks” herein and both terms meanpieces of dry or semi-moist pet food which can have a pellet shape orany other shape and can be made by slicing a food composition intoseparate pieces. Non-limiting examples of kibbles include particulates;pellets; pieces of pet food, dehydrated meat, meat analog, vegetables,and combinations thereof; and pet snacks, such as meat or vegetablejerky, rawhide, and biscuits. A “meat analog” is a meat emulsion productthat resembles pieces of natural meat in appearance, texture, andphysical structure.

The term “dietary supplement” means a product that is intended to beingested in addition to the normal animal diet. Dietary supplements maybe in any form, e.g., solid, liquid, gel, tablets, capsules, powder, andthe like. In one aspect, they can be provided in convenient dosageforms. In some embodiments, they can be provided in bulk consumerpackages such as bulk powders, liquids, gels, or oils. In otherembodiments, supplements can be provided in bulk quantities to beincluded in other food items such as snacks, treats, supplement bars,beverages and the like.

The compositions disclosed herein may lack any element that is notspecifically disclosed herein. Thus, a disclosure of an embodiment usingthe term “comprising” includes a disclosure of embodiments “consistingessentially of” and “consisting of” the components identified.Similarly, the methods disclosed herein may lack any step that is notspecifically disclosed herein. Thus, a disclosure of an embodiment usingthe term “comprising” includes a disclosure of embodiments “consistingessentially of” and “consisting of” the steps identified. Any embodimentdisclosed herein can be combined with any other embodiment disclosedherein unless explicitly and directly stated otherwise.

The present discussion of embodiments, aspects, examples, etc. areindependent in that they can apply to all methods and compositions. Forexample, a metabolite used in a pet food composition can also be used inthe method of modulating or a method of minimizing costs associated withmaking such a pet food, and vice versa.

Embodiments

In an aspect of the present disclosure, a pet food composition cancomprise protein, carbohydrates, fat, fiber, and a metabolite formodulating the amount of at least one of Bifidobacterium, Lactobacillus,or Clostridium perfringens in a companion animal, such as a senior dogor a senior cat. In one embodiment, the pet food composition can providean increase in the amount of at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens in the companion animal.

In another aspect of the present disclosure, a method of modulating theamount of at least one of Bifidobacterium, Lactobacillus, or Clostridiumperfringens in a companion animal is provided. The method can compriseadministering to the companion animal a pet food composition comprisingprotein, carbohydrates, fat, fiber, and a metabolite for modulating theamount of at least one of Bifidobacterium, Lactobacillus, or Clostridiumperfringens in a companion animal in the companion animal. In oneaspect, the companion animal can be a senior dog or a senior cat.

In still another aspect of the present disclosure, a method of measuringa change in the amount of at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens in a companion animal cancomprise obtaining a serum sample of the companion animal, measuringconcentrations of at least three distinct metabolites from the serumsample that modulate at least one of Bifidobacterium, Lactobacillus, orClostridium perfringens, and determining that the Bifidobacterium,Lactobacillus, or Clostridium perfringens has changed if, aftercomparing the metabolite concentrations to average metaboliteconcentrations of each metabolite of comparable companion animals, themetabolite concentrations are different than the average metaboliteconcentrations.

Yet another aspect of the present disclosure is a method of minimizingcosts associated with production of a pet food having a firstformulation designed for consumption by a companion animal, such as asenior dog or a senior cat. A further aspect of the present disclosureis a method of enhancing nutritional benefit of a pet food having afirst formulation designed for consumption by a companion animal, suchas a senior dog or a senior cat. These methods comprise adjusting thefirst formulation of the pet food to be a second formulation. At leastone of the first and second formulations comprises a metabolite formodulating the amount of Bifidobacterium, Lactobacillus, or Clostridiumperfringens in a companion animal. The adjusting comprises changing anamount of the metabolite in the first formulation to a different amountin the second formulation.

“Minimizing” costs means that the costs associated with making thesecond formulation are less than the costs associated with making thefirst formulation, for example on a per serving basis, per unit weight,per unit volume, per total energy, and the like. “Enhanced” nutritionalbenefit means that the nutritional benefit of the second formulation isgreater than the nutritional benefit of the first formulation.

As discussed herein, the pet food compositions and methods can contain ametabolite or multiple metabolites for modulating the amounts of variousbacterium. In various aspects, the compositions and methods can contain1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 metabolites for modulating theamount of any one of the bacterium, or multiple bacteria, or for eachbacterium. As such, in one embodiment, the composition can comprise atleast 6 metabolites, including metabolites for individually modulatingthe amount of each one of Bifidobacterium, Lactobacillus, andClostridium perfringens in a companion animal. In another embodiment,the composition can comprise at least 9 metabolites, including at least3 metabolites for individually modulating the amounts of each one ofBifidobacterium, Lactobacillus, and Clostridium perfringens in acompanion animal. In still another embodiment, the composition cancomprise at least 12 metabolites, including at least 4 metabolites forindividually modulating the amounts of each one of Bifidobacterium,Lactobacillus, and Clostridium perfringens in a companion animal.Additionally, in one embodiment, the composition can comprise at leastfour distinct metabolites that modulate the amounts of at least one ofBifidobacterium, Lactobacillus, or Clostridium perfringens. Further, inone embodiment, the composition can comprise at least five distinctmetabolites that modulate at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens. Still, in another embodiment,the method can modulate at least two bacterium selected from the groupconsisting of Bifidobacterium, Lactobacillus, and Clostridiumperfringens and where the composition comprises at least six distinctmetabolites, three distinct metabolites for modulating each of the atleast two bacterium. Yet, in another embodiment, the method can modulateBifidobacterium, Lactobacillus, and Clostridium perfringens and wherethe composition comprises at least nine distinct metabolites, threedistinct metabolites for modulating each of Bifidobacterium,Lactobacillus, and Clostridium perfringens. As previously noted, themetabolite and bacteria relationships can be used for both method andcomposition embodiments.

In some embodiments, the metabolite negatively modulates the specificbacterium, and the changing of the amount of the metabolite comprisesdecreasing the amount of the metabolite. In other embodiments, themetabolite negatively modulates the specific bacterium, and the changingof the amount of the metabolite comprises increasing the amount of themetabolite. In some embodiments, the metabolite positively modulates thespecific bacterium, and the changing of the amount of the metabolitecomprises increasing the amount of the metabolite. In other embodiments,the metabolite positively modulates the specific bacterium, and thechanging of the amount of the metabolite comprises decreasing the amountof the metabolite. These are not mutually exclusive embodiments; aparticular embodiment can comprise decreasing the amount of a metabolitethat negatively modulates a first specific bacterium and increasing theamount of a metabolite that positively modulates a second specificbacterium, and the first and second specific bacteria can be the same ordifferent specific bacteria (e.g., one or more of Bifidobacterium,Lactobacillus, or Clostridium perfringens).

Decreasing the amount of the metabolite can comprise decreasing theamount of the metabolite directly and/or decreasing the amount of aningredient which comprises the metabolite. In some embodiments,decreasing the amount of the metabolite can comprise decreasing theamount of a precursor of the metabolite directly and/or decreasing theamount of an ingredient which comprises a precursor of the metabolite.For example, the second formulation can contain, relative to the firstformulation, less of an ingredient having a high amount of themetabolite or precursor thereof (e.g., an ingredient having an amount ofthe metabolite or precursor thereof that is higher than in one or moreof the other ingredients).

Increasing the amount of the metabolite can comprise increasing theamount of the metabolite directly and/or decreasing the amount of aningredient which comprises the metabolite. In some embodiments,increasing the amount of the metabolite can comprise increasing theamount of a precursor of the metabolite directly and/or increasing theamount of an ingredient which comprises a precursor of the metabolite.For example, the second formulation can contain, relative to the firstformulation, more of an ingredient having a high amount of themetabolite or precursor thereof (e.g., an ingredient having an amount ofthe metabolite or precursor thereof that is higher than in one or moreof the other ingredients).

Generally, the methods and compositions described herein can provide anincrease or a decrease in the amount of at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens in the companion animal. Insome embodiment, the increase or decrease can be at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or even 50%. In one aspect, thecompositions and methods can provide an increase. In another aspect, thecompositions and methods can provide a decrease. As such, the presentmodulation, and resulting increase or decrease, can provide one of thefollowing health benefits: weight gain, increased feed conversion rate,reduced mortality, decreased digestive discomfort, relief from irritablebowel syndrome, relief from Crohn's disease, reduction in bacterialovergrowth, increase in food digestion, increase in lactose metabolism,decreased negative effects of antibiotics, healthy skin, decreased yeastinfections, increased absorption of nutrients, or increased immunesystem.

In one embodiment, the increase or decrease can be for at least two ofBifidobacterium, Lactobacillus, and Clostridium perfringens. In anotherembodiment, the increase or decrease can be for Bifidobacterium,Lactobacillus, and Clostridium perfringens.

Ingredients comprising the metabolite (e.g., a precursor of themetabolite) and optionally amounts of the metabolite in the ingredientcan be identified by analysis of the ingredient, for example using aseparation technique, such as gas chromatography or liquidchromatography, and then mass spectrometry.

In each of these compositions and methods, the pet food composition canbe a wet food, a semi-moist food or a dry food. In an embodiment, thepet food composition is one or more components of a blended composition.In some embodiments, the pet food composition is a kibble, and in someembodiments, the pet food composition is a meat analog. Additionally, inanother embodiment, the present composition for modulating a bacteriumcan be a dietary supplement comprising the metabolites described herein.Further, a method of modulating a bacterium can include administering tothe companion animal the dietary supplement.

Such pet food compositions can be administered to the companion animalin amounts ranging from about 3 g of pet food per 1 lb body weight toabout 16 g of pet food per 1 lb body weight of the companion animal.Additionally, the metabolites can be present in amounts from about 0.01weight % to about 10 weight % of the food composition. In one aspect,the metabolites can be present in concentrations of about 0.01 to about1,000 mg/kg of food. In another aspect, the metabolites can be presentin concentrations from about 1 IU to about 500,000 IU per kilogram offood. In one embodiment, the pet food composition can be administered tothe companion animal in amounts sufficient to maintain the health and/orbody weight of the animal. In one aspect, the administration can beregular administration.

As noted above and detailed later in this application, the presentinventors identified metabolite compounds which correlate toBifidobacterium, Lactobacillus, or Clostridium perfringens. Thus, themetabolite in the pet food composition can be one of these compounds.Nevertheless, the metabolite can be any metabolite for modulating theamount of at least one of Bifidobacterium, Lactobacillus, or Clostridiumperfringens in a companion animal, even if the metabolite is notexplicitly disclosed herein. For example, the metabolite can be acompound identified using the methods disclosed herein but not itselfexplicitly disclosed herein. Furthermore, the metabolite can be acompound identified using a method not disclosed herein if the compoundis reliably correlated to at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens.

As a non-limiting example, the metabolite can modulate Bifidobacteriumand can be selected from the group consisting of campesterol,7-ketodeoxycholate, 3-(4-hydroxyphenyl)propionate, 3-dehydrocarnitine,12-dehydrocholate, X-17314, X-15461, chenodeoxycholate,3-dehydrocholate, cholate, 15-methylpalmitate (isobar with2-methylpalmitate) and mixtures thereof. In one embodiment, themetabolite can modulate Bifidobacterium and can be selected from thegroup consisting of campesterol, 7-ketodeoxycholate,3-(4-hydroxyphenyl)propionate, 3-dehydrocarnitine, 12-dehydrocholate,chenodeoxycholate, 3-dehydrocholate, cholate, 15-methylpalmitate (isobarwith 2-methylpalmitate) and mixtures thereof.

As another non-limiting example, the metabolite can modulateLactobacillus and can be selected from the group consisting of X-15461,3-phenylpropionate (hydrocinnamate), 3-(4-hydroxyphenyl)propionate,mannitol, campesterol, indoleacetate, 3-dehydrocarnitine, X-11378 andmixtures thereof. In one embodiment, the metabolite can modulateLactobacillus and can be selected from the group consisting of3-phenylpropionate (hydrocinnamate), 3-(4-hydroxyphenyl)propionate,mannitol, campesterol, indoleacetate, 3-dehydrocarnitine, and mixturesthereof.

As yet another non-limiting example, the metabolite can modulateClostridium perfringens and can be selected from the group consisting ofdihomo-linoleate (20:2n6), arachidonate (20:4n6), adrenate (22:4n6),mannose, 3-hydroxybutyrate (BHBA), trigonelline (N′-methylnicotinate),eicosenoate (20:1n9 or 11), docosapentaenoate (n3 DPA; 22:5n3),palmitoyl sphingomyelin, glycerate, linolenate [alpha or gamma; (18:3n3or 6)], dihomo-linolenate (20:3n3 or n6), X-11378, X-17010,7-alpha-hydroxycholesterol, stearate (18:0), palmitate (16:0), glycolate(hydroxyacetate), stachydrine, X-12450, X-09789, campesterol,pantothenate, linoleate (18:2n6), docosapentaenoate (n6 DPA; 22:5n6),X-11412, X-17185, phenylalanine and mixtures thereof. In one embodiment,the metabolite can modulate Clostridium perfringens and can be selectedfrom the group consisting of dihomo-linoleate (20:2n6), arachidonate(20:4n6), adrenate (22:4n6), mannose, 3-hydroxybutyrate (BHBA),trigonelline (N′-methylnicotinate), eicosenoate (20:1n9 or 11),docosapentaenoate (n3 DPA; 22:5n3), palmitoyl sphingomyelin, glycerate,linolenate [alpha or gamma; (18:3n3 or 6)], dihomo-linolenate (20:3n3 orn6), 7-alpha-hydroxycholesterol, stearate (18:0), palmitate (16:0),glycolate (hydroxyacetate), stachydrine, campesterol, pantothenate,linoleate (18:2n6), docosapentaenoate (n6 DPA; 22:5n6), phenylalanineand mixtures thereof.

In yet another aspect of the present disclosure, a method enhancesnutritional benefit of a pet food having a first formulation designedfor consumption by companion animals, and the method comprisesadministering the pet food having the first formulation to a firstcompanion animal. The method further comprises measuring in a sample ofbody fluid from the companion animal (e.g., plasma) an amount of asurrogate marker comprising a metabolite that modulates at least one ofBifidobacterium, Lactobacillus, or Clostridium perfringens. The methodfurther comprises adjusting the first formulation of the pet food to bea second formulation having a difference from the first formulationselected from the group consisting of (i) an ingredient is present inthe second formulation and is absent in the first formulation, (ii) aningredient is absent in the second formulation and is present in thefirst formulation, (iii) an ingredient is present in the first andsecond formulations but in a different amount, and (iv) combinationsthereof. The adjusting is based at least partially on the amount of thesurrogate marker measured in the previous step.

The adjusting can comprise directly decreasing the amount of ametabolite negatively modulating a specific bacterium and/or decreasingthe amount of an ingredient which comprises a metabolite negativelymodulating a specific bacterium. In some embodiments, decreasing theamount of the metabolite can comprise decreasing the amount of aprecursor of the metabolite directly and/or decreasing the amount of aningredient which comprises a precursor of the metabolite. For example,the second formulation can contain, relative to the first formulation,less of an ingredient having a high amount of the metabolite orprecursor thereof (e.g., an ingredient having an amount of themetabolite or precursor thereof that is higher than in one or more ofthe other ingredients).

The adjusting can comprise increasing the amount of the metabolite cancomprise directly increasing the amount of a metabolite positivelymodulating a specific bacterium and/or increasing the amount of aningredient which comprises a metabolite positively modulating a specificbacterium. In some embodiments, increasing the amount of the metabolitecan comprise increasing the amount of a precursor of the metabolitedirectly and/or increasing the amount of an ingredient which comprises aprecursor of the metabolite. For example, the second formulation cancontain, relative to the first formulation, more of an ingredient havinga high amount of the metabolite or precursor thereof (e.g., aningredient having an amount of the metabolite or precursor thereof thatis higher than in one or more of the other ingredients).

As noted above, ingredients comprising the metabolite (e.g., a precursorof the metabolite) and optionally amounts of the metabolite in theingredient can be identified by analysis of the ingredient, for exampleusing a separation technique, such as gas chromatography or liquidchromatography, and then mass spectrometry.

The method further comprises producing the pet food in the secondformulation. In an embodiment, the method comprises administering thepet food having the second formulation to a second companion animal.

This method can be used to provide customized nutrition for a specificcompanion animal. For example, the first and second companion animal canbe the same specific companion animal such that the animal who isadministered the pet food having the first formulation has one or moreof their specific bacteria modulated by the first formulation assessed.Then this same animal is provided with the resultant second formulationwhich this animal will more effectively modulate the specific bacterium.Consequently, a pet owner can compensate for their pet's changes in oneor more of Bifidobacterium, Lactobacillus, or Clostridium perfringens.

Alternatively or additionally, this method can be used to providecustomized nutrition for companion animals who share one or more of agender, an approximate age, an approximate size (e.g., body weight,height, and/or length) or a breed. For example, the second companionanimal can be a different specific animal than the first companionanimal but has a characteristic selected from the group consisting of(i) about the same age as the first companion animal, (ii) about thesame size as the first companion animal, (iii) the same breed as thefirst companion animal, (iv) the same gender as the first companionanimal, and (iv) combinations thereof. Preferably, the second companionanimal is one of a plurality of companion animals who each share thecharacteristic with the first companion animal. The method can compriseadministering the pet food having the second formulation to theplurality of companion animals. In an embodiment, at least a portion ofthe plurality of companion animals is located remotely relative to thefirst companion animal.

The pet food compositions disclosed herein can be any food formulatedfor consumption by a pet such as a companion animal. In an embodiment,the pet food composition provides complete nutrition as defined by theAssociation of American Feed Control Officials (AAFCO) and which dependson the type of animal for which the composition is intended (e.g., dogor cat).

The pet food composition can comprise meat, such as emulsified meat.Examples of suitable meat include poultry, beef, pork, lamb and fish,especially those types of meats suitable for pets. The meat can includeany additional parts of an animal including offal. Some or all of themeat can be provided as one or more meat meals, namely meat that hasbeen dried and ground to form substantially uniform-sized particles andas defined by AAFCO. Additionally or alternatively, vegetable proteincan be used, such as pea protein, corn protein (e.g., ground corn orcorn gluten), wheat protein (e.g., ground wheat or wheat gluten), soyprotein (e.g., soybean meal, soy concentrate, or soy isolate), riceprotein (e.g., ground rice or rice gluten) and the like.

The pet food compositions disclosed herein can comprise vegetable oil, aflavorant, a colorant and water. Suitable vegetable oils include soybeanoil, corn oil, cottonseed oil, sunflower oil, canola oil, peanut oil,safflower oil, and the like. Examples of suitable flavorants includeyeast, tallow, rendered animal meals (e.g., poultry, beef, lamb, pork),flavor extracts or blends (e.g., grilled beef), animal digests, and thelike. Suitable colorants include FD&C colors, such as blue no. 1, blueno. 2, green no. 3, red no. 3, red no. 40, yellow no. 5, yellow no. 6,and the like; natural colors, such as caramel coloring, annatto,chlorophyllin, cochineal, betanin, turmeric, saffron, paprika, lycopene,elderberry juice, pandan, butterfly pea and the like; titanium dioxide;and any suitable food colorant known to the skilled artisan.

The pet food compositions disclosed herein can optionally includeadditional ingredients, such as other grains and/or other starchesadditionally or alternatively to flour, amino acids, fibers, sugars,animal oils, aromas, other oils additionally or alternatively tovegetable oil, humectants, preservatives, polyols, salts, oral careingredients, antioxidants, vitamins, minerals, probiotic microorganisms,bioactive molecules or combinations thereof.

Suitable starches include a grain such as corn, rice, wheat, barley,oats, soy and the like, and mixtures of these grains, and can beincluded at least partially in any flour. Suitable humectants includesalt, sugars, propylene glycol and polyhydric glycols such as glycerinand sorbitol, and the like. Suitable oral care ingredients includealfalfa nutrient concentrate containing chlorophyll, sodium bicarbonate,phosphates (e.g., tricalcium phosphate, acid pyrophosphates, tetrasodiumpyrophosphate, metaphosphates, and orthophosphates), peppermint, cloves,parsley, ginger and the like. Examples of suitable antioxidants includebutylated hydroxyanisole (“BHA”) and butylated hydroxytoluene (“BHT”),vitamin E (tocopherols), and the like.

Non-limiting examples of vitamins that can be used include Vitamins A,B-complex (such as B-1, B-2, B-6 and B-12), C, D, E and K, niacin andacid vitamins such as pantothenic acid and folic acid and biotin.Non-limiting examples of suitable minerals include calcium, iron, zinc,magnesium, iodine, copper, phosphorus, manganese, potassium, chromium,molybdenum, selenium, nickel, tin, silicon, vanadium, boron and thelike.

Non-limiting examples of suitable preservatives include potassiumsorbate, sorbic acid, sodium methyl para-hydroxybenzoate, calciumpropionate, propionic acid, and combinations thereof.

Specific amounts for each additional ingredient in the pet foodcompositions disclosed herein will depend on a variety of factors suchas the ingredient included in the first edible material and any secondedible material; the species of animal; the animal's age, body weight,general health, sex, and diet; the animal's consumption rate; thepurpose for which the food product is administered to the animal; andthe like. Therefore, the components and their amounts may vary widely.

For example, the amount of any of the above-noted ingredients can bedecreased or increased based on the estimated effect on the amount ofone or more of Bifidobacterium, Lactobacillus, or Clostridiumperfringens (e.g., an effect identified by one of the methods disclosedherein). In an embodiment, the amount of one or more of the above-notedingredients can be increased if such ingredients comprise a metabolitethat positively modulates one or more of Bifidobacterium, Lactobacillus,or Clostridium perfringens. Additionally or alternatively, the amount ofone or more of the above-noted ingredients can be decreased if suchingredients comprise a metabolite that negatively modulates one or moreof Bifidobacterium, Lactobacillus, or Clostridium perfringens.

As noted above, ingredients comprising the metabolite (e.g., a precursorof the metabolite) and optionally amounts of the metabolite in theingredient can be identified by analysis of the ingredient, for exampleusing a separation technique, such as gas chromatography or liquidchromatography, and then mass spectrometry.

EXAMPLES

The following non-limiting examples are illustrative of embodiments ofthe present disclosure.

Methods

Each of the examples was derived from the following study.

83 Canines were all fed Diet A for 5 weeks, followed by a 1 weektransition period and then 15 were fed Diet B for 5 weeks as shown inthe Table 1 below. Plasma samples were taken after overnight fastingusing EDTA vacutainer tubes during the fifth week of feeding of eachdiet. After centrifugation, plasma was aliquoted into cryovials andfrozen at −80° C. Fecal samples were used to determine levels ofbacteria. Levels of Bifidobacterium, Lactobacillus, or Clostridiumperfringens were determined based on published 16S sequences usingquantitative PCR.

TABLE 1 Moisture Moisture Basis % DM % Protein % Fat % Ash % Fiber % CHO% GE kcal/g Diet A As-Is 8.1 91.9 22.7 13.3 6.1 2.0 47.9 4.5 Dry 0 10024.7 14.5 6.6 2.1 52.1 4.9 matter Diet B As-Is 76 24 9.1 10.5 1.8 0 2.61.7 Dry 0 100 38 43.7 7.5 0 10.8 6.9 Matter

Metabolomic analysis was carried out using the following methods byMetabolon Inc. Samples were extracted and split into equal parts foranalysis on GC/MS and LC/MS/MS platforms. Proprietary software was usedto match ions to an in-house library of standards for metaboliteidentification and for metabolite quantitation by peak area integrationby Metabolon Inc. Mass and retention index are provided in the followingtables such that each metabolite can be uniquely identified andindividually distinguished.

At the time of analysis, samples were thawed and extracts prepared toremove protein, dislodge small molecules bound to protein or physicallytrapped in the precipitated protein matrix, and recover a wide range ofchemically diverse metabolites. A separate aliquot of each experimentalplasma sample was taken then pooled for the creation of “Client Matrix”(CMTRX) samples. These CMTRX samples were injected throughout theplatform run and served as technical replicates, allowing variability inthe quantitation of all consistently detected biochemicals to bedetermined and overall process variability and platform performance tobe monitored. Extracts of all experimental and CMTRX samples were splitfor analysis on the GC/MS and LC/MS/MS platforms.

The CMTRX technical replicate samples were treated independentlythroughout the process as if they were client study samples. All processsamples (CMTRX and Grob test mixtures of organic components used toassess GC column performance, process blanks, etc.) were spaced evenlyamong the injections for each day and all client samples were randomlydistributed throughout each day's run. Data were collected over multipleplatform run days and thus ‘block normalized’ by calculating the medianvalues for each run-day block for each individual compound. Thisnormalization minimizes any inter-day instrument gain or drift, but doesnot interfere with intra-day sample variability. Missing values (if any)were assumed to be below the level of detection for that biochemicalwith the instrumentation used and were imputed with the observed minimumfor that particular biochemical.

A number of internal standards were added to each experimental andprocess standard sample just prior to injection into the massspectrometers. A measure of the platform variability (7%) was determinedby calculating the median relative standard deviation (RSD) for theseinternal standards. Because these standards are added to the samplesimmediately prior to injection into the instrument, this value reflectsinstrument variation. In addition, the median relative standarddeviation (RSD) for the biochemicals that were consistently measured inthe CMTRX represents the total variability within the process for theactual experimental samples and the variability in quantitation of theendogenous metabolites within these samples (12%). Results for the CMTRXand internal standards indicated that the platform produced data thatmet process specifications.

589 total metabolites were detected in plasma. This total corresponds tomany biochemicals (401) that matched a named structure in the referencelibrary (named compounds). The remaining biochemicals (188) representdistinct chemical entities (that is, they represent a single molecule ofdiscrete molecular formula and structure), but they do not currentlymatch a named biochemical in the reference library (unnamed/unknowncompounds).

Example 1 (Clostridium perfringens)

Metabolite correlations with Clostridium perfringens were determinedbased on plasma metabolomics (Table 2). This provided a predictive modelof compounds which can influence Clostridium perfringens eitherpositively or negatively. Feeding different levels of these compounds(diet B vs diet A; Table 3), and noting changes in Clostridiumperfringens (Table 4), served as a validation model. The metabolitecompositions of the two different diets were determined to identifyrelative levels of specific compounds. Those validated by the model areshown in Table 5.

TABLE 2 Metabolite correlations with Clostridium perfringens.Correlations with a P value < 0.01 are reported. Correlation RetentionID Correlation P-Value Index Mass beta-hydroxyisovalerate 0.53891.24E−07 1043 117.1 X-18559 0.5374 1.36E−07 1864.9 254.1dihomo-linoleate (20:2n6) 0.535 1.58E−07 5722 307.3 X-11949 0.52013.97E−07 3830 220.1 arachidonate (20:4n6) 0.5143 5.61E−07 5525 303.4X-12672 0.5116 6.56E−07 1797 256 adrenate (22:4n6) 0.5003 1.26E−06 5684331.3 mannose 0.4832 3.22E−06 1753.5 203.9 3-hydroxybutyrate (BHBA)0.4818 3.48E−06 1203.5 116.9 X-11787 −0.4742 5.19E−06 1126 148.1trigonelline (N′-methylnicotinate) −0.4657 8.03E−06 757 138.1cysteine-glutathione disulfide −0.4638 8.85E−06 821 427.1 eicosenoate(20:1n9 or 11) 0.4606 1.04E−05 5955 309.4 4-vinylphenol sulfate −0.44761.96E−05 3323 199.1 docosapentaenoate (n3 DPA; 22:5n3) 0.4442 2.31E−055574 329.4 2-aminobutyrate 0.434 3.73E−05 758 104.1 alpha-tocopherol0.4248 5.65E−05 2305.4 502.5 X-16009 0.4173 7.85E−05 2191 222 palmitoylsphingomyelin 0.4126 9.60E−05 2524 311.3 pipecolate 0.4083 0.0001 1120130.1 glycerate 0.4085 0.0001 1360.7 189 cysteine −0.4069 0.0001 1560.1218 X-11549 0.4037 0.0001 5093 339.3 linolenate [alpha or gamma; (18:3n3or 6)] 0.4006 0.0002 5450 277.3 dihomo-linolenate (20:3n3 or n6) 0.38420.0003 5600 305.4 betaine 0.3832 0.0003 721 118.2 X-11378 0.3853 0.00035325 445.4 hydroquinone sulfate 0.3861 0.0003 1383 1891,5-anhydroglucitol (1,5-AG) −0.375 0.0004 1788.7 217 X-11437 −0.37840.0004 2888 231 glutarylcarnitine (C5) 0.3684 0.0006 1565 276.1 X-16015−0.3577 0.0008 3788 268.1 X-17010 0.3544 0.0009 3169.5 189 X-16869−0.3563 0.0009 5179.8 330.2 2-linoleoylglycerophosphoethanolamine −0.3520.001 5650 476.4 stearoyl sphingomyelin 0.3464 0.0012 #N/A #N/A X-124350.3471 0.0012 3174 357.2 7-alpha-hydroxycholesterol 0.3444 0.0013 2300456.2 1-oleoylglycerophosphoethanolamine −0.3425 0.0014 5928 478.3X-13543 −0.3423 0.0014 1096 484.1 stearate (18:0) 0.3414 0.0015 5886283.4 palmitate (16:0) 0.3395 0.0016 5619 255.3 X-13866 −0.3396 0.00162406 253.1 X-12339 0.3371 0.0017 1055 174.1 glycolate (hydroxyacetate)0.3376 0.0017 1119 177 X-18570 −0.3381 0.0017 3243.1 207 arginine 0.32920.0022 728 173.2 threonate 0.3228 0.0027 1560.7 292.1 stachydrine−0.3222 0.0028 860 144.1 catechol sulfate −0.3191 0.0031 1928 188.9X-12450 0.3179 0.0032 5397 251.4 2-ethylhexanoate 0.3146 0.0036 1210.5201.2 X-09789 −0.3143 0.0036 2613 153.1 X-18558 0.3128 0.0038 1676.8380.1 campesterol −0.3088 0.0043 2353 343.4 pantothenate 0.3055 0.00472218 220.1 linoleate (18:2n6) 0.2992 0.0057 5533 279.3 docosapentaenoate(n6 DPA; 22:5n6) 0.2979 0.0059 5625 329.42-oleoylglycerophosphoethanolamine −0.2973 0.006 5848 478.3 X-114120.2975 0.006 3836 204.2 X-16120 0.2968 0.0061 1271.7 164.1 X-17185−0.2937 0.0067 3069.1 215.2 X-17502 −0.2886 0.0078 5093.4 297.31-palmitoylglycerophosphoinositol 0.288 0.0079 #N/A #N/A X-18487 −0.28770.008 1269.6 273.1 phenylalanine −0.2869 0.0081 2056 166.11-linoleoylglycerophosphoethanolamine −0.2829 0.0091 5725 476.3 X-11843−0.2817 0.0094 2710 230.1 2-hydroxybutyrate (AHB) 0.28 0.0099 1169.4130.9

TABLE 3 Compound levels present in diets A and B. Values representscaled imputed values (data are scaled such that the median valuemeasured across all samples was set to 1.0 and missing values (if any)were imputed with the observed minimum for that particular compound). IDA B beta-hydroxyisovalerate 0.48 0.48 X-18559 0.78 0.78 dihomo-linoleate(20:2n6) 0.33 4.06 X-11949 0.28 0.28 arachidonate (20:4n6) 0.43 4.48X-12672 0.72 0.72 adrenate (22:4n6) 0.82 5.31 mannose 0.21 1.453-hydroxybutyrate (BHBA) 0.18 0.34 X-11787 0.04 0.06 trigonelline(N′-methylnicotinate) 12.47 1.65 cysteine-glutathione disulfide 0.040.04 eicosenoate (20:1n9 or 11) 0.32 2.11 4-vinylphenol sulfate 0.020.02 docosapentaenoate (n3 DPA; 22:5n3) 0.14 4.84 2-aminobutyrate 1.580.83 alpha-tocopherol 0.01 0.03 X-16009 0.2 0.2 palmitoyl sphingomyelin0.03 0.2 pipecolate 7.41 1.56 glycerate 3.38 9.94 cysteine 0.21 0.24X-11549 0.13 0.13 linolenate [alpha or gamma; (18:3n3 or 6)] 0.83 1.66dihomo-linolenate (20:3n3 or n6) 0.42 5.03 betaine 0.61 0.69 X-113780.05 0.11 hydroquinone sulfate 0.03 0.03 1,5-anhydroglucitol (1,5-AG)0.28 0.28 X-11437 0.05 0.05 glutarylcarnitine (C5) 0.27 0.27 X-160150.02 0.02 X-17010 0.68 2.51 X-16869 0.29 0.292-linoleoylglycerophosphoethanolamine 0.56 2.58 stearoyl sphingomyelin#N/A #N/A X-12435 0.36 0.36 7-alpha-hydroxycholesterol 0.74 4.911-oleoylglycerophosphoethanolamine 0.16 5.65 X-13543 0.1 0.1 stearate(18:0) 0.33 1.26 palmitate (16:0) 0.4 0.88 X-13866 0.26 0.26 X-123390.96 0.5 glycolate (hydroxyacetate) 0.89 0.94 X-18570 0.08 0.08 arginine1 0.63 threonate 0.61 0.22 stachydrine 3.62 0.78 catechol sulfate 0.020.02 X-12450 0.17 1.43 2-ethylhexanoate 0.06 0.02 X-09789 5.54 0.2X-18558 0.55 0.47 campesterol 1.56 0.72 pantothenate 12.83 19.07linoleate (18:2n6) 0.8 0.92 docosapentaenoate (n6 DPA; 22:5n6) 0.61 4.772-oleoylglycerophosphoethanolamine 0.43 6.03 X-11412 0.13 0.21 X-161200.45 0.45 X-17185 0.12 0.06 X-17502 0.42 1.151-palmitoylglycerophosphoinositol #N/A #N/A X-18487 0.26 0.26phenylalanine 0.75 0.64 1-linoleoylglycerophosphoethanolamine 0.48 2.77X-11843 0.19 0.19 2-hydroxybutyrate (AHB) 0.28 0.1

TABLE 4 Clostridium perfringens in response to diet A and Diet B.Difference Diet A Diet B p- Parameter Mean StdDev Mean StdDev MeanStdErr value Clostridium 5.6 1.1 8.2 0.4 −2.6528 0.2948 0 perfringens

TABLE 5 Compounds validated by dietary change. ID RI Massdihomo-linoleate (20:2n6) 5722 307.3 arachidonate (20:4n6) 5525 303.4adrenate (22:4n6) 5684 331.3 Mannose 1753.5 203.9 3-hydroxybutyrate(BHBA) 1203.5 116.9 trigonelline (N′-methylnicotinate) 757 138.1eicosenoate (20:1n9 or 11) 5955 309.4 docosapentaenoate (n3 DPA; 22:5n3)5574 329.4 palmitoyl sphingomyelin 2524 311.3 Glycerate 1360.7 189linolenate [alpha or gamma; (18:3n3 or 6)] 5450 277.3 dihomo-linolenate(20:3n3 or n6) 5600 305.4 X-11378 5325 445.4 X-17010 3169.5 1897-alpha-hydroxycholesterol 2300 456.2 stearate (18:0) 5886 283.4palmitate (16:0) 5619 255.3 glycolate (hydroxyacetate) 1119 177stachydrine 860 144.1 X-12450 5397 251.4 X-09789 2613 153.1 campesterol2353 343.4 pantothenate 2218 220.1 linoleate (18:2n6) 5533 279.3docosapentaenoate (n6 DPA; 22:5n6) 5625 329.4 X-11412 3836 204.2 X-171853069.1 215.2 phenylalanine 2056 166.1

Example 2 (Lactobacillus)

Metabolite correlations with Lactobacillus were determined based onplasma metabolomics (Table 6). This provided a predictive model ofcompounds which can influence Lactobacillus either positively ornegatively. Feeding different levels of these compounds (diet B vs dietA; Table 7), and noting changes in Lactobacillus (Table 8), served as avalidation model. The metabolite compositions of the two different dietswere determined to identify relative levels of specific compounds. Thosecompounds validated by the model are shown in Table 9.

TABLE 6 Metabolite correlations with Lactobacillus. Correlations with aP value < 0.01 are reported. Correlation Retention ID CorrelationP-Value Index Mass X-15461 0.5878 4.15E−09 2125 160.1 equol sulfate0.5318 1.93E−07 3625 321.2 X-12156 0.5251 2.92E−07 3238 233.1indolepropionate 0.5218 3.59E−07 1925 202.1 3-phenylpropionate(hydrocinnamate) 0.4995 1.32E−06 2830 149.1 X-11334 0.4939 1.80E−06 982259.1 3-(4-hydroxyphenyl)propionate 0.4715 6.00E−06 1727.9 179.1mannitol 0.4548 1.38E−05 1839 319.1 campesterol 0.4416 2.62E−05 2353343.4 indoleacrylate 0.433 3.89E−05 2529 186.1 indoleacetate 0.43044.38E−05 3760 176.1 indoleacetylglutamine 0.4286 4.76E−05 2530 302.21-arachidonoylglycerophosphoethanolamine 0.4205 6.81E−05 5731 500.3X-12212 0.4198 7.03E−05 3607 229.1 X-12236 0.4166 8.09E−05 1321 245.1X-17443 0.4068 0.0001 3102.3 271.2 1-oleoylglycerophosphoethanolamine0.3838 0.0003 5928 478.3 catechol sulfate 0.3844 0.0003 1928 188.93-dehydrocarnitine 0.3706 0.0005 1020 160.2 X-15636 0.3581 0.0008 3814243.1 2-arachidonoylglycerophosphoethanolamine 0.3526 0.001 5674 500.3X-15728 0.352 0.001 3972 231.1 2-oleoylglycerophosphoethanolamine 0.35020.0011 5848 478.3 taurolithocholate 0.3485 0.0012 5328 482.43-(3-hydroxyphenyl)propionate 0.3471 0.0012 2000 165.11-linoleoylglycerophosphoethanolamine 0.3434 0.0014 5725 476.34-acetylphenol sulfate 0.3421 0.0014 2399 2151-palmitoylplasmenylethanolamine 0.3424 0.0014 6153 436.4N4-acetylcytidine 0.3336 0.0019 2030 286.11-heptadecanoylglycerophosphoethanolamine 0.3315 0.0021 6029 466.3succinylcarnitine 0.3286 0.0023 1401 262.1 X-11378 −0.3281 0.0023 5325445.4 octadecanedioate 0.3133 0.0037 5033 313.3 N-acetylornithine 0.30920.0042 875 175.2 X-16015 0.3089 0.0043 3788 268.1 X-12010 0.3065 0.00461707 203.1 N-acetyltryptophan −0.305 0.0048 2650 245.2 kynurenine 0.29770.006 1902 209.1 N-acetyltyrosine −0.2967 0.0061 1677 222.2 adenosine5′-monophosphate (AMP) 0.2927 0.0069 #N/A #N/A X-11987 −0.2894 0.00761409 139.2 X-16480 0.2872 0.0081 4685.5 309.3 benzoate 0.285 0.00861291.5 179

TABLE 7 Compound levels present in diets A and B. Values representscaled imputed values (data are scaled such that the median valuemeasured across all samples was set to 1.0 and missing values (if any)were imputed with the observed minimum for that particular compound). IDA B X-15461 0.97 0.35 equol sulfate 0.08 0.08 X-12156 0.19 0.19indolepropionate 0.07 0.07 3-phenylpropionate (hydrocinnamate) 18.680.13 X-11334 0.19 0.19 3-(4-hydroxyphenyl)propionate 14.94 0.38 mannitol12.51 1.72 campesterol 1.56 0.72 indoleacrylate 0.06 0.06 indoleacetate1.78 0.46 indoleacetylglutamine 0.52 0.521-arachidonoylglycerophosphoethanolamine 0.14 4.6 X-12212 0.21 0.21X-12236 0.1 0.1 X-17443 0.21 0.21 1-oleoylglycerophosphoethanolamine0.16 5.65 catechol sulfate 0.02 0.02 3-dehydrocarnitine 0.54 0.34X-15636 0.2 0.2 2-arachidonoylglycerophosphoethanolamine 0.33 10.33X-15728 0.32 0.32 2-oleoylglycerophosphoethanolamine 0.43 6.03taurolithocholate 0.45 2.42 3-(3-hydroxyphenyl)propionate 0.1 0.11-linoleoylglycerophosphoethanolamine 0.48 2.77 4-acetylphenol sulfate0.4 0.56 1-palmitoylplasmenylethanolamine 0.21 3.23 N4-acetylcytidine0.53 0.53 1-heptadecanoylglycerophosphoethanolamine 0.18 2.28succinylcarnitine 0.32 0.32 X-11378 0.05 0.11 octadecanedioate 0.06 0.07N-acetylornithine 0.41 0.21 X-16015 0.02 0.02 X-12010 0.5 0.5N-acetyltryptophan 1 0.24 kynurenine 0.05 0.04 N-acetyltyrosine 0.820.32 adenosine 5′-monophosphate (AMP) #N/A #N/A X-11987 0.14 0.14X-16480 0.06 0.06 benzoate 0.03 0.03

TABLE 8 Lactobacillus in response to diet A and Diet B. Difference DietA Diet B p- Parameter Mean StdDev Mean StdDev Mean StdErr valueLactobacillus 8.6 1.1 5.8 1.9 2.8 0.6 0

TABLE 9 Compounds validated by dietary change. Retention ID Index MassX-15461 2125 160.1 3-phenylpropionate (hydrocinnamate) 2830 149.13-(4-hydroxyphenyl)propionate 1727.9 179.1 mannitol 1839 319.1campesterol 2353 343.4 indoleacetate 3760 176.1 3-dehydrocarnitine 1020160.2 X-11378 5325 445.4

Example 3 (Bifidobacterium)

Metabolite correlations with Bifidobacterium were determined based onplasma metabolomics (Table 10). This provided a predictive model ofcompounds which can influence Bifidobacterium either positively ornegatively. Feeding different levels of these compounds (diet B vs dietA; Table 11), and noting changes in Bifidobacterium (Table 12), servedas a validation model. The metabolite compositions of the two differentdiets were determined in order to identify relative levels of specificcompounds. Those compounds validated by the model are shown in Table 13.

TABLE 10 Metabolite correlations with Bifidobacterium. Correlations witha P value < 0.01 are reported. Correlation Retention ID CorrelationP-Value Index Mass indoleacrylate 0.5948 2.43E−09 2529 186.1 equolsulfate 0.5634 2.41E−08 3625 321.2 X-12212 0.5426 9.74E−08 3607 229.1indolepropionate 0.5355 1.54E−07 1925 202.1 X-15636 0.5102 7.14E−07 3814243.1 X-15728 0.4442 2.31E−05 3972 231.1 X-11541 0.4292 4.62E−05 4984399 X-16015 0.4259 5.37E−05 3788 268.1 campesterol 0.4223 6.30E−05 2353343.4 X-12236 0.4159 8.35E−05 1321 245.11-linoleoylglycerophosphoethanolamine 0.3896 0.0002 5725 476.32-oleoylglycerophosphoethanolamine 0.3987 0.0002 5848 478.3 catecholsulfate 0.3949 0.0002 1928 188.9 4-acetylphenol sulfate 0.385 0.00032399 215 ornithine 0.3772 0.0004 1763.8 141.9 7-ketodeoxycholate 0.37740.0004 4840 405.4 10-heptadecenoate (17:1n7) 0.3752 0.0004 5558 267.3pentadecanoate (15:0) 0.3728 0.0005 1853.5 299.2 carnosine 0.3625 0.0007813 225.2 3-(4-hydroxyphenyl)propionate 0.3591 0.0008 1727.9 179.1benzoate 0.3525 0.001 1291.5 179 3-dehydrocarnitine 0.3491 0.0011 1020160.2 12-dehydrocholate 0.3411 0.0015 4866 405.4 X-17612 0.3402 0.00154298.5 567.4 3-(3-hydroxyphenyl)propionate 0.3278 0.0023 2000 165.1cis-vaccenate (18:1n7) 0.3262 0.0025 1987 339.3 X-18570 0.3258 0.00253243.1 207 X-17314 0.3244 0.0026 2595 266.2 X-11843 0.3248 0.0026 2710230.1 hydroquinone sulfate −0.3247 0.0026 1383 189 X-15461 0.3135 0.00372125 160.1 2-arachidonoylglycerophosphoethanolamine 0.312 0.0039 5674500.3 X-11852 0.3043 0.0049 3324 233.1 chenodeoxycholate 0.3025 0.00525250 391.4 myristoleate (14:1n5) 0.301 0.0054 5338 225.33-dehydrocholate 0.2938 0.0067 5050 405.4 cholate 0.2933 0.0068 5148407.4 X-11909 0.2925 0.0069 5272 297.3 X-12668 0.2912 0.0072 2318 246.1myristate (14:0) 0.291 0.0073 5439 227.3 deoxycholate 0.2882 0.0079 5268391.4 oleate (18:1n9) 0.2867 0.0082 1984.4 339.2 15-methylpalmitate(isobar with 2- 0.283 0.0091 5698 269.4 methylpalmitate)phenylalanyltryptophan 0.2818 0.0094 3349 352.2 N-acetyltyrosine −0.28130.0095 1677 222.2

TABLE 11 Compound levels present in diets A and B. Values representscaled imputed values (data are scaled such that the median valuemeasured across all samples was set to 1.0 and missing values (if any)were imputed with the observed minimum for that particular compound). IDA B indoleacrylate 0.06 0.06 equol sulfate 0.08 0.08 X-12212 0.21 0.21indolepropionate 0.07 0.07 X-15636 0.2 0.2 X-15728 0.32 0.32 X-115410.36 0.36 X-16015 0.02 0.02 campesterol 1.56 0.72 X-12236 0.1 0.11-linoleoylglycerophosphoethanolamine 0.48 2.772-oleoylglycerophosphoethanolamine 0.43 6.03 catechol sulfate 0.02 0.024-acetylphenol sulfate 0.4 0.56 ornithine 4.31 15.34 7-ketodeoxycholate2.65 0.38 10-heptadecenoate (17:1n7) 0.09 0.33 pentadecanoate (15:0)0.34 0.7 carnosine 0.38 1.28 3-(4-hydroxyphenyl)propionate 14.94 0.38benzoate 0.03 0.03 3-dehydrocarnitine 0.54 0.34 12-dehydrocholate 0.220.17 X-17612 0.21 0.3 3-(3-hydroxyphenyl)propionate 0.1 0.1cis-vaccenate (18:1n7) 0.32 0.73 X-18570 0.08 0.08 X-17314 0.42 0.13X-11843 0.19 0.19 hydroquinone sulfate 0.03 0.03 X-15461 0.97 0.352-arachidonoylglycerophosphoethanolamine 0.33 10.33 X-11852 0.1 0.1chenodeoxycholate 7.78 0.79 myristoleate (14:1n5) 0.12 0.413-dehydrocholate 0.8 0.39 cholate 25.83 0.83 X-11909 0.16 0.2 X-126680.13 0.13 myristate (14:0) 0.15 0.4 deoxycholate 0.19 0.19 oleate(18:1n9) 0.34 1 15-methylpalmitate (isobar with 2-methylpalmitate) 0.370.19 phenylalanyltryptophan 0.29 0.29 N-acetyltyrosine 0.82 0.32

TABLE 12 Bifidobacterium in response to diet A and Diet B. DifferenceDiet A Diet B p- Parameter Mean StdDev Mean StdDev Mean StdErr valueBifidobacterium 7.0 1.3 4.4 1.1 2.5 0.5 0

TABLE 13 Compounds validated by dietary change. Retention ID Index Masscampesterol 2529 186.1 7-ketodeoxycholate 3625 321.23-(4-hydroxyphenyl)propionate 3607 229.1 3-dehydrocarnitine 1925 202.112-dehydrocholate 3814 243.1 X-17314 3972 231.1 X-15461 4984 399chenodeoxycholate 3788 268.1 3-dehydrocholate 2353 343.4 cholate 1321245.1 15-methylpalmitate (isobar with 2-methylpalmitate) 5725 476.3

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

The invention is claimed as follows:
 1. A pet food composition,comprising: protein, carbohydrates, fat, fiber, and at least 6 distinctmetabolites for individually modulating the amount of Bifidobacterium,Lactobacillus, and Clostridium perfringens in a companion animal;wherein the metabolite that modulates Bifidobacterium is selected fromthe group consisting of campesterol, 7-ketodeoxycholate,3-(4-hydroxyphenyl)propionate, 3-dehydrocarnitine, 12-dehydrocholate,X-17314, X-15461, chenodeoxycholate, 3-dehydrocholate, cholate,15-methylpalmitate (isobar with 2-methylpalmitate) and mixtures thereof;wherein the metabolite that modulates Lactobacillus is selected from thegroup consisting of X-15461, 3-phenylpropionate (hydrocinnamate),3-(4-hydroxyphenyl)propionate, mannitol, campesterol, indoleacetate,3-dehydrocarnitine, X-11378 and mixtures thereof; wherein the metabolitethat modulates Clostridium perfringens is selected from the groupconsisting of dihomo-linoleate (20:2n6), arachidonate (20:4n6), adrenate(22:4n6), mannose, 3-hydroxybutyrate (BHBA), trigonelline(N′-methylnicotinate), eicosenoate (20:1n9 or 11), docosapentaenoate (n3DPA; 22:5n3), palmitoyl sphingomyelin, glycerate, linolenate [alpha orgamma; (18:3n3 or 6)], dihomo-linolenate (20:3n3 or n6), X-11378,X-17010, 7-alpha-hydroxycholesterol, stearate (18:0), palmitate (16:0),glycolate (hydroxyacetate), stachydrine, X-12450, X-09789, campesterol,pantothenate, linoleate (18:2n6), docosapentaenoate (n6 DPA; 22:5n6),X-11412, X-17185, phenylalanine and mixtures thereof; and wherein thepet food composition provides at least a 5% increase or a 5% decrease inthe amount of at least one of Bifidobacterium, Lactobacillus, andClostridium perfringens in the companion animal upon administration ofthe pet food to the companion animal.
 2. The pet food composition ofclaim 1, wherein the composition comprises at least 9 distinctmetabolites, including at least 3 distinct metabolites for modulatingeach one of Bifidobacterium, Lactobacillus, and Clostridium perfringensin a companion animal.
 3. The pet food composition of claim 1, whereinthe composition comprises at least 12 distinct metabolites, including atleast 4 distinct metabolites for modulating each one of Bifidobacterium,Lactobacillus, and Clostridium perfringens in a companion animal.
 4. Thepet food composition of claim 1, wherein the metabolite that modulatesBifidobacterium is selected from the group consisting of campesterol,7-ketodeoxycholate, 3-(4-hydroxyphenyl)propionate, 3-dehydrocarnitine,12-dehydrocholate, and mixtures thereof.
 5. The pet food composition ofclaim 1, wherein the metabolite that modulates Lactobacillus is selectedfrom the group consisting of X-15461, 3-phenylpropionate(hydrocinnamate), 3-(4-hydroxyphenyl)propionate, mannitol, and mixturesthereof.
 6. The pet food composition of claim 1, wherein the metabolitethat modulates Clostridium perfringens is selected from the groupconsisting of dihomo-linoleate (20:2n6), arachidonate (20:4n6), adrenate(22:4n6), mannose, 3-hydroxybutyrate (BHBA), trigonelline(N′-methylnicotinate), eicosenoate (20:1n9 or 11), docosapentaenoate (n3DPA; 22:5n3), palmitoyl sphingomyelin, glycerate, linolenate [alpha orgamma; (18:3n3 or 6)], dihomo-linolenate (20:3n3 or n6), X-11378,X-17010, and mixtures thereof.
 7. The pet food composition of claim 1,wherein the pet food composition provides at least a 10% increase in theamount of at least one of Bifidobacterium, Lactobacillus, or Clostridiumperfringens in the companion animal.
 8. The pet food composition ofclaim 1, wherein the pet food composition provides at least a 10%decrease in the amount of at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens in the companion animal.
 9. Amethod of modulating the amount of at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens in a companion animal,comprising: administering to the companion animal a pet food compositioncomprising protein, carbohydrates, fat, fiber, and at least threedistinct metabolites for modulating the amount of at least one ofBifidobacterium, Lactobacillus, or Clostridium perfringens in thecompanion animal; wherein the metabolite that modulates Bifidobacteriumis selected from the group consisting of campesterol,7-ketodeoxycholate, 3-(4-hydroxyphenyl)propionate, 3-dehydrocarnitine,12-dehydrocholate, X-17314, X-15461, chenodeoxycholate,3-dehydrocholate, cholate, 15-methylpalmitate (isobar with2-methylpalmitate) and mixtures thereof; wherein the metabolite thatmodulates Lactobacillus is selected from the group consisting ofX-15461, 3-phenylpropionate (hydrocinnamate), 3-(4-hydroxyphenyl)propionate, mannitol, campesterol, indoleacetate, 3-dehydrocarnitine,X-11378 and mixtures thereof; and wherein the metabolite that modulatesClostridium perfringens is selected from the group consisting ofdihomo-linoleate (20:2n6), arachidonate (20:4n6), adrenate (22:4n6),mannose, 3-hydroxybutyrate (BHBA), trigonelline (N′-methylnicotinate),eicosenoate (20:1n9 or 11), docosapentaenoate (n3 DPA; 22:5n3),palmitoyl sphingomyelin, glycerate, linolenate [alpha or gamma; (18:3n3or 6)], dihomo-linolenate (20:3n3 or n6), X-11378, X-17010,7-alpha-hydroxycholesterol, stearate (18:0), palmitate (16:0), glycolate(hydroxyacetate), stachydrine, X-12450, X-09789, campesterol,pantothenate, linoleate (18:2n6), docosapentaenoate (n6 DPA; 22:5n6),X-11412, X-17185, phenylalanine and mixtures thereof.
 10. The method ofclaim 9, wherein the composition comprises at least four distinctmetabolites for modulating at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens.
 11. The method of claim 9,wherein the composition comprises at least five distinct metabolites formodulating at least one of Bifidobacterium, Lactobacillus, orClostridium perfringens.
 12. The method of claim 9, wherein the methodmodulates at least two bacteria selected from the group consisting ofBifidobacterium, Lactobacillus, and Clostridium perfringens and whereinthe composition comprises at least six distinct metabolites, threedistinct metabolites for modulating each of the at least two bacteria.13. The method of claim 9, wherein the method modulates Bifidobacterium,Lactobacillus, and Clostridium perfringens and wherein the compositioncomprises at least nine distinct metabolites, three distinct metabolitesfor modulating each of Bifidobacterium, Lactobacillus, and Clostridiumperfringens.
 14. The method of claim 9, wherein the pet food compositionprovides at least a 5% increase or a 5% decrease in the amount of atleast one of Bifidobacterium, Lactobacillus, or Clostridium perfringensin the companion animal.
 15. The method of claim 9, wherein theadministering is a regular administration.
 16. The method of claim 9,wherein the modulating provides one of the following health benefits:weight gain, increased feed conversion rate, reduced mortality,decreased digestive discomfort, relief from irritable bowel syndrome,relief from Crohn's disease, reduction in bacterial overgrowth, increasein food digestion, increase in lactose metabolism, decreased negativeeffects of antibiotics, healthy skin, decreased yeast infections,increased absorption of nutrients, or increased immune system.
 17. Amethod of measuring a change in the amount of at least one ofBifidobacterium, Lactobacillus, or Clostridium perfringens in acompanion animal, comprising: obtaining a serum sample of the companionanimal; measuring concentrations of at least three distinct metabolitesfrom the serum sample that modulate at least one of Bifidobacterium,Lactobacillus, or Clostridium perfringens; and determining that theBifidobacterium, Lactobacillus, or Clostridium perfringens has changedif, after comparing the metabolite concentrations to average metaboliteconcentrations of each metabolite from comparable companion animals, themetabolite concentrations are different than the average metaboliteconcentrations; wherein the metabolite that modulates Bifidobacterium isselected from the group consisting of campesterol, 7-ketodeoxycholate,3-(4-hydroxyphenyl)propionate, 3-dehydrocarnitine, 12-dehydrocholate,X-17314, X-15461, chenodeoxycholate, 3-dehydrocholate, cholate,15-methylpalmitate (isobar with 2-methylpalmitate) and mixtures thereof;wherein the metabolite that modulates Lactobacillus is selected from thegroup consisting of X-15461, 3-phenylpropionate (hydrocinnamate),3-(4-hydroxyphenyl)propionate, mannitol, campesterol, indoleacetate,3-dehydrocarnitine, X-11378 and mixtures thereof; and wherein themetabolite that modulates Clostridium perfringens is selected from thegroup consisting of dihomo-linoleate (20:2n6), arachidonate (20:4n6),adrenate (22:4n6), mannose, 3-hydroxybutyrate (BHBA), trigonelline(N′-methylnicotinate), eicosenoate (20:1n9 or 11), docosapentaenoate (n3DPA; 22:5n3), palmitoyl sphingomyelin, glycerate, linolenate [alpha orgamma; (18:3n3 or 6)], dihomo-linolenate (20:3n3 or n6), X-11378,X-17010, 7-alpha-hydroxycholesterol, stearate (18:0), palmitate (16:0),glycolate (hydroxyacetate), stachydrine, X-12450, X-09789, campesterol,pantothenate, linoleate (18:2n6), docosapentaenoate (n6 DPA; 22:5n6),X-11412, X-17185, phenylalanine and mixtures thereof.
 18. The method ofclaim 17, measuring concentrations of at least four distinct metabolitesfor modulating at least one of Bifidobacterium, Lactobacillus, orClostridium perfringens.
 19. The method of claim 17, measuringconcentrations of at least five distinct metabolites for modulating atleast one of Bifidobacterium, Lactobacillus, or Clostridium perfringens.20. The method of claim 17, wherein the metabolite that modulatesBifidobacterium is selected from the group consisting of campesterol,7-ketodeoxycholate, 3-(4-hydroxyphenyl)propionate, 3-dehydrocarnitine,12-dehydrocholate, and mixtures thereof; wherein the metabolite thatmodulates Lactobacillus is selected from the group consisting ofX-15461, 3-phenylpropionate (hydrocinnamate),3-(4-hydroxyphenyl)propionate, mannitol, and mixtures thereof; orwherein the metabolite that modulates Clostridium perfringens isselected from the group consisting of dihomo-linoleate (20:2n6),arachidonate (20:4n6), adrenate (22:4n6), mannose, 3-hydroxybutyrate(BHBA), trigonelline (N′-methylnicotinate), eicosenoate (20:1n9 or 11),docosapentaenoate (n3 DPA; 22:5n3), palmitoyl sphingomyelin, glycerate,linolenate [alpha or gamma; (18:3n3 or 6)], dihomo-linolenate (20:3n3 orn6), X-11378, X-17010, and mixtures thereof.